Insect pests are a major factor causing the loss of yield of the agricultural crops, and reducing the loss caused by insect pests is an important way to increase the yield and quality of food and feed crops. According to statistics, the loss caused by insect pest attacks on the total yield of food and feed crops in the world has been up to 14% every year, and has directly caused economic losses as high as hundreds of billions of US dollars to agricultural production. Use of the control measures, such as spraying chemical pesticides and biological insecticides, may do reduce the damage of pests to agricultural crops, but chemical pesticides cause environmental pollution, and biological insecticides are expensive. For a long time, spraying large amounts of chemical pesticides will not only enhance pests' resistance to pesticides, and damage beneficial insects and other ecosystems, but also seriously pollute the environments, increase the production costs, and destroy the ecological balance. Therefore, reducing the usage amount of insecticides and developing modern biotechnology has become a problem that must be faced in sustainable development of agriculture.
Maize is an important feed and industrial crop. At present, maize pests mainly include corn borers, which cause serious attacking and significant reduction of maize yield. Therefore, taking effective measures to control the damage thereof is of important significance to improving the maize yield and increasing the rural income. Damage caused by corn borers is one of the important biohazards resulting in reduction of maize yield throughout the year, and seriously affects the output and quality of maize. The corn borers include Ostrinia furnacalis and Ostrinia nubilalis. China is a district where maize is frequently and seriously attacked by Ostrinia furnacalis, and such an attack on a large scale occurs almost once every two years. The yield of maize is reduced by 10% to 15% by corn borers attack in the years of general situation, and may be reduced by as high as 30% or more or even total crop failure in the years under large-scale attack. Due to the damage caused by the corn borers, the loss of maize yield reaches 6-9 million tons every year. The corn borers not only directly cause the loss of maize yield, but also may induce and aggravate the occurrence of maize ear rot, thereby reducing the quality of maize.
At present, the corn borers are still controlled mainly by insecticides and pesticides. An insect-resistant Bt gene may be introduced into maize species using transgenic technology, thus improving the insect resistance of transgenic maize, reducing the usage amount of pesticides, and saving labors, material resources and social resources.
A Bt gene-encoded insecticidal crystal protein from Bacillus thuringiensis (Bt for short) is a Gram-positive soil bacillus. In the process of sporulation, Bt produces an insecticidal parasporal crystal protein known as δ-endotoxin. The protein has a very high insecticidal activity. The action principle thereof is that this insect-resistant protein can be dissolved by an alkaline intestinal juice and hydrolyzed to smaller active toxin fragments—the core fragments (Hofte and Whiteley, 1989). Further hydrolysis of the core fragments by protease can be avoided, and the activated proteins bond with the brush vesicles on the intestinal tracts of insects, resulting in perforation and further affecting osmotic balance. Cells expand and then are dissolved. Target organisms stop ingestion and finally die. Researches have shown that the intestinal epithelial cells of many Bt protein-targeted pests have highly affinitive binding sites (Hofte and Whiteley, 1989). Over the past few decades, dozens of Bacillus thuringiensis and more than 130 insecticidal crystal proteins encoded by them have been identified. Researches have proven that the Bt crystal protein is nonhazardous for human bodies, mammals, birds, fishes and a lot of beneficial insects, and does not pollute the environment. Therefore, Bt preparations have been applied in agriculture, forestry and environmental health as a nonhazardous natural microbial pesticide for nearly 50 years.
The Bt crystal protein must be ingested by insects to exert the function of killing the insects. However, the Bt crystal protein has poor stability in the natural environment; its insecticidal effect is greatly affected by the weather; it is readily degraded after exposure to sunlight; it cannot penetrate into plant tissues; and it is easily washed away by rain and dew. These factors greatly limit its development and application.
Vaeck et al. (Nature 328: 3-37, 1987) obtained a transgenic Bt insect-resistant tobacco having an insecticidal protein for the first time, from which weak insect resistance could be detected, and the expressed protein thereof was almost undetectable, only accounting for 0.001% of the soluble protein. Willbur et al. (Plant Physiol. 92: 1-11, 1990) demonstrated through researches that there was a significant difference in the use of codons between low grade biological bacteria and high-grade plants. In addition, there has been evidence that the mRNA transcribed from unstable ATTTA, AATAA or other sequences of tRNA in plants is not complete, so that the translated protein is too short to have an insecticidal activity.